This invention is related to the field of cancer diagnosis and treatment. In particular it is related to the use of gene expression to categorize and detect tumors.
The building of large databases containing human genome sequences is the basis for studies of gene expressions in various tissues during normal physiological and pathologic conditions. Constantly (constitutively) expressed sequences as well as sequences whose expression is altered during disease processes are important for our understanding of cellular properties, and for the identification of candidate genes for future therapeutic intervention. As the number of known genes and ESTs build up in the databases, array-based simultaneous screening of thousands of genes is necessary to obtain a profile of transcriptional behaviour, and to identify key genes that, either alone or in combination with other genes, control various aspects of cellular life. One cellular behaviour that has been a mystery for many years is the malignant behaviour of cancer cells. We now know that, for example, defects in DNA repair can lead to cancer, but the cancer-creating mechanism in heterozygous individuals is still largely unknown, as is the malignant cell""s ability to repeat cell cycles, to avoid apoptosis, to escape the immune system, to invade and metastasize, and to escape therapy. There are hints and indications in these areas and excellent progress has been made, but the myriad of genes interacting with each other in a highly complex multidimensional network is making the road to insight long and contorted.
Similar appearing tumorsxe2x80x94morphologically, histochemically, microscopicallyxe2x80x94can be profoundly different. They can have different invasive and metastasizing properties, as well as respond differently to therapy. There is a thus a need in the art for methods which distinguish tumors and tissues on different bases than are currently in use in the clinic.
It is an object of the present invention to provide a method of determining an expression pattern of a cell sample independent of the proportion of submucosal, smooth muscle, or connective tissue cells present.
It is another object of the present invention to provide a method of determining an expression pattern of a cell sample.
It is an object of the present invention to provide a method for determining an expression pattern of a urothelium or bladder cancer cell.
Another object of the invention is to provide a method of detecting an invasive tumor in a patient.
Another object of the invention is to provide a method to diagnose a bladder cancer.
Another object of the invention is to provide a method to predict outcome or prescribe treatment of a bladder tumor.
Another object of the invention is to provide a method to determine grade or stage of a bladder tumor.
Still another object of the invention is to provide a method of identifying a tissue sample as urothelial.
Yet another object of the invention provides a method of determining an expression pattern of a bladder tissue sample independent of the proportion of submucosal, muscle, and connective tissue cells present.
These and other objects of the invention are achieved by providing one or more of the embodiments described below. In one embodiment a method is provided of determining an expression pattern of a cell sample independent of the proportion of submucosal, smooth muscle, or connective tissue cells present. Expression is determined of one or more genes in a sample comprising cells. The one or more genes exclude genes which are expressed in the submucosal, muscle, and connective tissue. A pattern of expression is formed for the sample which is independent of the proportion of submucosal, muscle, and connective tissue cells in the sample.
In another aspect of the invention a method of determining an expression pattern of a cell sample is provided. Expression is determined of one or more genes in a sample comprising cells. A first pattern of expression is thereby formed for the sample. Genes which are expressed in submucosal, smooth muscle, or connective tissue cells are removed from the first pattern of expression, forming a second pattern of expression which is independent of the proportion of submucosal, smooth muscle, or connective tissue cells in the sample.
Another embodiment of the invention provides a method for determining an expression pattern of a urothelium or bladder cancer cell. Expression is determined of one or more genes in a sample comprising urothelium or bladder cancer cells; the expression determined forms a first pattern of expression. A second pattern of expression which was formed using the one or more genes and a sample comprising predominantly submucosal, smooth muscle, or connective tissue cells, is subtracted from the first pattern of expression, forming a third pattern of expression. The third pattern of expression reflects expression of the urothelium or bladder cancer cells independent of the proportion of submucosal, smooth muscle, or connective tissue cells present in the sample.
In another embodiment of the invention a method is provided of detecting an invasive tumor in a patient. A marker is detected in a sample of a body fluid. The body fluid is selected from the group consisting of blood, plasma, serum, urine, ascites fluid, pleural fluid, spinal fluid, sputum, and mucous secretions. The marker is an mRNA or protein expression product of a gene which is more prevalent in submucosal, smooth muscle, or connective tissue than in the body fluid. An increased amount of the marker in the body fluid indicates a tumor which has become invasive in the patient.
In another aspect of the invention a method is provided for diagnosing a bladder cancer. A first pattern of expression is determined of one or more genes in a bladder tissue sample suspected of being neoplastic. The first pattern of expression is compared to a second and third reference pattern of expression. The second pattern is of the one or more genes in normal urothelium and the third pattern is of the one or more genes in bladder cancer. A first pattern of expression which is found to be more similar to the third pattern than the second indicates neoplasia of the bladder tissue sample.
According to yet another aspect of the invention a method is provided for predicting outcome or prescribing treatment of a bladder tumor. A first pattern of expression is determined of one or more genes in a bladder tumor sample. The first pattern is compared to one or more reference patterns of expression determined for bladder tumors at a grade between I and IV. The reference pattern which shares maximum similarity with the first pattern is identified. The outcome or treatment appropriate for the grade of tumor of the reference pattern with the maximum similarity is assigned to the bladder tumor sample.
In another embodiment of the invention a method is provided for determining grade of a bladder tumor. A first pattern of expression is determined of one or more genes in a bladder tumor sample. The first pattern is compared to one or more reference patterns of expression determined for bladder tumors at a grade between I and IV. The reference pattern which shares maximum similarity with the first pattern is identified. The grade of the reference pattern with the maximum similarity is assigned to the bladder tumor sample.
Yet another embodiment of the invention provides a method to determine stage of a bladder tumor. A first pattern of expression is determined of one or more genes in a bladder tumor sample. The first pattern is compared to one or more reference patterns of expression determined for bladder tumors at different stages. The reference pattern which shares maximum similarity with the first pattern is identified. The stage of the reference pattern with the maximum similarity is assigned to the bladder tumor sample.
In still another embodiment of the invention a method is provided for identifying a tissue sample as urothelial. A first pattern of expression is determined of one or more genes in a tissue sample. The first pattern of expression is compared to a second pattern of expression obtained from normal urothelial cells. Similarity between the first and second patterns identifies the tissue sample is urothelial in its origin.
Another aspect of the invention is a method to identify a set of genes useful for diagnosing, predicting outcome, or prescribing treatment of a bladder cancer. A first pattern of expression is determined of one or more genes in a first bladder tissue sample. A second pattern of expression is determined of the one or more genes in a second bladder tissue sample. The first bladder tissue sample is a normal urothelium sample or an earlier stage or lower grade of bladder tumor than the second bladder tissue sample. The first pattern of expression is compared to the second pattern of expression to identify a first set of genes whose expression is increased or decreased in the second bladder tissue sample relative to the first bladder tissue sample. Those genes which are expressed in submucosal, smooth muscle or connective tissue are removed from the first set of genes to produce a second set of genes. Expression of the second set of genes can be used for diagnosing, predicting outcome, or prescribing treatment of a bladder cancer.
According to yet another aspect of the invention a method is provided for determining an expression pattern of a bladder tissue sample independent of the proportion of submucosal, smooth muscle, or connective tissue cells present. A single-cell suspension of disaggregated bladder tumor cells is isolated from a bladder tissue sample comprising bladder cells, submucosal cells, smooth muscle cells, or connective tissue cells. The expression of one or more genes in the single-cell suspension is determined. A pattern of expression is thus formed for the sample which is independent of the proportion of submucosal, smooth muscle, or connective tissue cells in the bladder tissue sample.
According to still another aspect of the invention a method is provided for screening compounds to identify candidate therapeutic agents for treating bladder cancer. Bladder tumor cells are contacted with a test compound. Gene expression of one or more genes is determined in the bladder tumor cells which have been contacted with the test compound. The one or more genes are ones whose expression changes during the development of a bladder cancer. A test compound is identified as a candidate therapeutic agent if it causes gene expression of at least one of the one or more genes to change to a level which is characteristic of an earlier stage of cancer progression.
The present invention thus provides the art with numerous methods for molecularly assessing bladder cells. The methods aid the art in diagnosing, identifying, classifying, treating, detecting, and treating tumors of the bladder.